GENEKAL DEVELOPMENT OF A LOCALIZATION PROGRAM 527 



onstration of a direct effect on the coupling process. In addition to this, 

 if the uncoupling is to be made the basis for the effects observed on living 

 cells or tissues, evidence must be presented that the action seen in vitro 

 actually occurs within the cell and that it is the primary action. 



GENERAL DEVELOPMENT OF A LOCALIZATION PROGRAM 



Some of the procedures frequently useful in the localization of the site 

 of action of an inhibitor have been discussed and it is now possible to take 

 up the problem of the over-all design of the investigation. Although it is 

 true that each new inhibitor will usually present a problem that is at least 

 in some respects unique and will require an individual approach, it is pos- 

 sible to formulate some principles of general applicability. The total pro- 

 gram may be divided for convenience into four chronological phases: (1) 

 obtaining the preliminary data, (2) planning the attack, (3) sequential anal- 

 lysis for the inhibition site, and (4) correlation of the action at this site 

 with the original effects observed. 



The Initial Phase 



A substance has been found to have an action on living tissue, either 

 isolated or in the whole organism, that is indicative of an interference with 

 metabolism. There are certain things it is often useful to do before starting 

 or even planning a more detailed analysis of the site of action. Easily ob- 

 tained results at this stage will often save considerable time later. It is 

 suggested that two basic types of data be obtained initially. 



(1) A dose or concentration relationship should be established, preferably 

 over the entire range of action of the substance. This will not only provide 

 information on the concentrations to be used in the later work and a basis 

 for comparison of the in vitro and in vivo results, but occasionally will 

 bring out features of the action that will aid in the localization. 



A simple example of this may be cited. It w^as observed originally that 

 low concentrations (0.01-0.03 mM) of 1,10-phenanthroline blocked almost 

 completely the oxidation of pyruvate and a-ketoglutarate by heart mito- 

 chondria, but during the process of obtaining a concentration-inhibition 

 curve it was found that higher concentrations (0.1-0.3 mM) did not inhibit 

 as strongly, and actually stimulated the oxygen uptake in some instances 

 (Yang et al, 1958). This unexpected result pointed to certain characteristics 

 of the inhibition and later suggested experiments that led to the demonstra- 

 tion of an uncoupling action on the phosphorylation associated with the 

 oxidation of DPNH. However, the basic reason for the reversal of inhibi- 

 tion has not been elucidated and any postulated mechanism in the future 

 must be able to explain this reversal. 



